A key regulator of both the Rho GTPase GDP/GTP activity cycle and the cytosol/membrane localization cycle is RhoGDI. Our current understanding of RhoGDI regulation and the dynamics of its interactions with Rho GTPases during in vivo cell signaling remains largely hypothetical, based upon in vitro biochemical observations. We have recently shown phosphorylation to be a key regulator of the Rho GTPase-RhoGDI cycle. We propose to develop new biosensor and computational approaches to elucidate the intracellular dynamics and regulation of the Rho GTPase cycle in the context of integrin, nephrin, and Angiotensin II signaling. These studies will thus provide basic information about Rho GTPase regulation relevant to cardiovascular and/or renal function. Fluorescent biosensors of RhoGDI binding to Cdc42, and of Src activation will be developed. These biosensors will pioneer new, generally applicable approaches, Adjusting wavelengths of dyes will enable us to produce complementary biosensor pairs that can be imaged in the same cell with subsecond and submicron resolution (Cdc42-GDI binding with Cdc42 activation or Src activation). The data will be analyzed with a computational tool, Virtual Biosensor, that will model the imaging experiments in the context of the cell physiology. Solution of this "forward problem" will permit us to derive in vivo concentrations and rates. Key steps in the proposed GTPase regulatory cycle will be investigated in vivo to quantitatively evaluate the protein-protein interactions, kinetics, and coordination of Rho GTPase regulation by RhoGDI. Complex formation/dissociation will be related to overall cycle dynamics, to regulatory phosphorylations, to Rho GTPase activation, and to the resulting biological (ie. cytoskeletal) outcomes. Regulation of Rho GTPase- RhoGDI complexes through Src-mediated phosphorylation will be evaluated, making use of the novel Src kinase and GDI complexation biosensors in vivo, combined with simultaneous imaging techniques, to relate kinase activation and phosphorylation of RhoGDI to the dynamics of Rho GTPase cycling. Quantitative analysis with new "Virtual Microscopy" tools will be applied to imaging data to derive parameters for kinetic models that can be further tested through biochemical and genetic manipulations. Computational modeling and simulation will permit hypothesis testing to assess whether the key steps in the pathway have been identified and correctly characterized. PUBLIC HEALTH RELEVANCE: There is substantial evidence that Rho GTPases and their critical regulatory protein Rho GDI play important roles in both normal physiology and in disease. Understanding the basic dynamics of how RhoGDI regulates Rho GTPase activity and cytosol-to- membrance cycling in live cells is critical to understanding the function of Rho GTPases in biology. With our studies, we hope to provide a quantitatively detailed, predictive model that can be used to investigate Rho GTPase function in health and disease.